Mutations conferring SO42− pumping ability on the cyanobacterial anion pump rhodopsin and the resultant unique features of the mutant

Membrane transport proteins can be divided into two types: those that bind substrates in a resting state and those that do not. In this study, we demonstrate that these types can be converted by mutations through a study of two cyanobacterial anion-pumping rhodopsins, Mastigocladopsis repens halorhodopsin (MrHR) and Synechocystis halorhodopsin (SyHR). Anion pump rhodopsins, including MrHR and SyHR, initially bind substrate anions to the protein center and transport them upon illumination. MrHR transports only smaller halide ions, Cl- and Br-, but SyHR also transports SO42−, despite the close sequence similarity to MrHR. We sought a determinant that could confer SO42− pumping ability on MrHR and found that the removal of a negative charge at the anion entrance is a prerequisite for SO42− transport by MrHR. Consistently, the reverse mutation in SyHR significantly weakened SO42− pump activity. Notably, the MrHR and SyHR mutants did not show SO42− induced absorption spectral shifts or changes in the photoreactions, suggesting no bindings of SO42− in their initial states or the bindings to the sites far from the protein centers. In other words, unlike wild-type SyHR, these mutants take up SO42− into their centers after illumination and release it before the ends of the photoreactions.

This difference is detected as a voltage difference between the two ITO electrodes. 6 Figure S5. Cl --induced absorption spectral shifts of the wild-type MrHR (a) and the E182T/F-helix mutant (b). The medium contained 50 mM citric acid (pH 6.0), 0.1% DDM and NaCl (0 -400 mM).
The shift directions are indicated with black arrows. The absorbance changes at 538 nm (A538) were plotted in (c) and were fitted with the following equation: where Kd, [Cl -], and y0 represent the dissociation constant, Clconcentration, and the absorbance value in the absence of Cl -, respectively. The determined Kd values are indicated in the panel.
7 Figure S6. Absorption spectra of the E. coli membrane fragments containing wild-type MrHR (a) and the E182T/F-helix mutant (b). The basal buffer was 50 mM citric acid, pH 6.0. In addition to the bands at approximately 500-600 nm, narrow bands from cytochrome appeared at approximately 410 nm. The latter peak values were used to normalize the respective spectra.
8 Figure S7. Light minus dark difference spectra of wild-type MrHR (a-e) and the E182T/F-helix mutant (f-j). The flash-induced absorbance changes were measured at 400-700 nm with 10 nm intervals. These data were used to calculate the difference spectra. The samples and the measuring conditions were the same as those in Fig. 6. In the presence of Cl -(d, e, i, j), both proteins exhibited small spectral changes in the early time range (< 500 μs). These changes are plotted in the insets of the respective panels.

Validity of the method for estimating the Clpump amounts in E. coli cells
In this study, relative expressions of Clpumps were estimated by the magnitudes of the flash-induced absorbance changes at 540 nm of the E. coli lysates (see Materials and Methods). 540 nm is a wavelength close to λmax of MrHR and SyHR. Thus, the magnitudes of the negatively deflected signals at 540 nm reflect the expression amounts of the Clpumps. However, this method requires the assumption that mutations do not significantly distort the photochemical properties of Clpumps, including the quantum efficiencies of the photoreactions, the photoreactions themselves, and the extinction coefficients of dark states and intermediates. This assumption seems reasonable. This is because when we measured the Clpump activities of the wild-types and mutant Clpumps, we observed light-induced pH changes of similar magnitude (Fig. 2). To emphasize the validity of this method, we confirmed that there are no significant distortions in the photoreactions and the molar extinction coefficients in the dark states for representative mutants. Figure S10 compares the flash-induced absorbance changes among the wild-type proteins and the representative mutants. For these measurements, we prepared the acrylamide gels containing E. coli membrane fragments expressing the respective proteins. Thus, Fig. S10 shows the results of a separate experiment from the estimation of protein expression amounts. In Fig. S10, time-courses of absorbance changes at three selected wavelengths are shown in the respective left panels. For calculations of the relative protein amounts, we picked up the maximum values of negatively deflected signals at 540 nm, whose time points are indicated by the vertical broken lines in the left panels. The respective right panels show the difference spectra at those time points. All the difference spectra have nearly the same shapes, indicating that the photoreactions are nearly identical, at least at those time points. Figure S11 and Table S1 are the experimental results of obtaining the molar extinction coefficients (ε) in the dark state. Here, we prepared the purified proteins and measured the absorption spectra before and after the bleach reaction by hydroxylamine (HA). The black lines in Fig Table S1. Differences from wild-type protein values were within ±2.8% for MrHR mutants and within ±4.6% for SyHR mutants. These results also indicate that there are no significant distortions of photochemical properties due to mutations.  The ε values were calculated from the spectra in Supplementary Fig. S11 according to the following equation: = 33,900 × -HA +HA where 33,900 is the ε value of the retinal oxime, A-HA and A+HA represent the absorbance peaks of rhodopsin and retinal oxime at around 536 nm and 360 nm, respectively.